Bottom Line:
Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms.Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends.These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin.

ABSTRACTMegakaryocytes release mature platelets in a complex process. Platelets are known to be released from intermediate structures, designated proplatelets, which are long, tubelike extensions of the megakaryocyte cytoplasm. We have resolved the ultrastructure of the megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy. Platelet production begins with the extension of large pseudopodia that use unique cortical bundles of microtubules to elongate and form thin proplatelet processes with bulbous ends; these contain a peripheral bundle of microtubules that loops upon itself and forms a teardrop-shaped structure. Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms. Microtubule coils similar to those observed in blood platelets are detected only at the ends of proplatelets and not within the platelet-sized beads found along the length of proplatelet extensions. Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends. These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin. We propose that mature platelets are assembled de novo and released only at the ends of proplatelets, and that the complex bending and branching observed during proplatelet morphogenesis represents an elegant mechanism to increase the numbers of proplatelet ends.

Figure 9: Model of platelet production suggested by these experiments and previous studies. After commitment to the megakaryocyte lineage, cells undergo polyploidization and cytoplasmic maturation (Stage 1). During the initial stages of proplatelet formation, megakaryocytes remodel their cytoplasm into thick pseudopodia (Stage 2), which contain bundles of microtubules situated just beneath the protruding membrane. Blunt pseudopodia elongate into proplatelet processes, which harbor thick bundles of microtubules in their core and contain a teardrop-shaped loop at their distal tip (Stage 3). Proplatelets frequently bend and form a branched structure from which new processes extend (Stage 4). These proplatelet processes form constrictions along their length giving the beaded appearance to proplatelets (Stage 5). The swellings along proplatelets are unstable structures presumed to contain packets of platelet material in the process of being delivered to the ends. Proplatelets are released from the megakaryocyte body after a retraction (Stage 6), and may undergo further fragmentation to yield individual platelets.

Mentions:
Here, we report detailed studies aimed at investigating discrete steps in platelet formation by terminally differentiated mouse megakaryocytes, and our analysis represents the first effort to dissect dynamic aspects of this process. Proplatelets extend by a microtubule-based system, bifurcate repeatedly to increase the number of ends, and deliver packets of platelet material to these ends. Proplatelets and their contents are highly dynamic structures that continuously engage in alternate extension and retraction, reversible spreading, and bidirectional transport of particles. This dynamic behavior is inconsistent with the notion of proplatelets as static, linear arrays of nascent blood platelets, and strongly suggests that platelets mature along and at the ends of proplatelets in the final stages of an elaborate process. Our analysis suggests the model for platelet formation shown in Fig. 9.

Figure 9: Model of platelet production suggested by these experiments and previous studies. After commitment to the megakaryocyte lineage, cells undergo polyploidization and cytoplasmic maturation (Stage 1). During the initial stages of proplatelet formation, megakaryocytes remodel their cytoplasm into thick pseudopodia (Stage 2), which contain bundles of microtubules situated just beneath the protruding membrane. Blunt pseudopodia elongate into proplatelet processes, which harbor thick bundles of microtubules in their core and contain a teardrop-shaped loop at their distal tip (Stage 3). Proplatelets frequently bend and form a branched structure from which new processes extend (Stage 4). These proplatelet processes form constrictions along their length giving the beaded appearance to proplatelets (Stage 5). The swellings along proplatelets are unstable structures presumed to contain packets of platelet material in the process of being delivered to the ends. Proplatelets are released from the megakaryocyte body after a retraction (Stage 6), and may undergo further fragmentation to yield individual platelets.

Mentions:
Here, we report detailed studies aimed at investigating discrete steps in platelet formation by terminally differentiated mouse megakaryocytes, and our analysis represents the first effort to dissect dynamic aspects of this process. Proplatelets extend by a microtubule-based system, bifurcate repeatedly to increase the number of ends, and deliver packets of platelet material to these ends. Proplatelets and their contents are highly dynamic structures that continuously engage in alternate extension and retraction, reversible spreading, and bidirectional transport of particles. This dynamic behavior is inconsistent with the notion of proplatelets as static, linear arrays of nascent blood platelets, and strongly suggests that platelets mature along and at the ends of proplatelets in the final stages of an elaborate process. Our analysis suggests the model for platelet formation shown in Fig. 9.

Bottom Line:
Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms.Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends.These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin.

ABSTRACTMegakaryocytes release mature platelets in a complex process. Platelets are known to be released from intermediate structures, designated proplatelets, which are long, tubelike extensions of the megakaryocyte cytoplasm. We have resolved the ultrastructure of the megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy. Platelet production begins with the extension of large pseudopodia that use unique cortical bundles of microtubules to elongate and form thin proplatelet processes with bulbous ends; these contain a peripheral bundle of microtubules that loops upon itself and forms a teardrop-shaped structure. Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms. Microtubule coils similar to those observed in blood platelets are detected only at the ends of proplatelets and not within the platelet-sized beads found along the length of proplatelet extensions. Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends. These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin. We propose that mature platelets are assembled de novo and released only at the ends of proplatelets, and that the complex bending and branching observed during proplatelet morphogenesis represents an elegant mechanism to increase the numbers of proplatelet ends.